U.S. patent application number 14/127298 was filed with the patent office on 2015-01-08 for methods and systems for tracking bioremediation processes.
This patent application is currently assigned to Arizona State Board of Regents For and on Behalf of Arizona State University. The applicant listed for this patent is Rolf U. Halden, Rosa Krajmalnik-Brown, James W. Wilson. Invention is credited to Rolf U. Halden, Rosa Krajmalnik-Brown, James W. Wilson.
Application Number | 20150010945 14/127298 |
Document ID | / |
Family ID | 47423175 |
Filed Date | 2015-01-08 |
United States Patent
Application |
20150010945 |
Kind Code |
A1 |
Krajmalnik-Brown; Rosa ; et
al. |
January 8, 2015 |
METHODS AND SYSTEMS FOR TRACKING BIOREMEDIATION PROCESSES
Abstract
A method for expression of engineered constructs containing
sequences coding for functional genes of interest in E. coli
strains. Here tceA and tceB are fused to an inducible, active
promoter to optimize transcription of the open reading frames, and
an introduced consensus Shine-Dalgarno sequence allowing for
optimal ribosome binding and translation of the open reading
frames, and codons optimized for expression in E. coil. This
over-expressed protein is then used to design appropriate mass
spectro metric methods for environmental detection.
Inventors: |
Krajmalnik-Brown; Rosa;
(Chandler, AZ) ; Halden; Rolf U.; (Phoenix,
AZ) ; Wilson; James W.; (Drexel Hill, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Krajmalnik-Brown; Rosa
Halden; Rolf U.
Wilson; James W. |
Chandler
Phoenix
Drexel Hill |
AZ
AZ
PA |
US
US
US |
|
|
Assignee: |
Arizona State Board of Regents For
and on Behalf of Arizona State University
Scottsdale
AZ
|
Family ID: |
47423175 |
Appl. No.: |
14/127298 |
Filed: |
June 19, 2012 |
PCT Filed: |
June 19, 2012 |
PCT NO: |
PCT/US12/43174 |
371 Date: |
March 20, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61501020 |
Jun 24, 2011 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
250/282; 435/320.1; 435/471 |
Current CPC
Class: |
C12N 15/63 20130101;
C12N 15/67 20130101; C12N 15/70 20130101; C07K 14/195 20130101;
H01J 49/04 20130101; H01J 49/0031 20130101 |
Class at
Publication: |
435/69.1 ;
435/471; 435/320.1; 250/282 |
International
Class: |
C12N 15/70 20060101
C12N015/70; H01J 49/00 20060101 H01J049/00; H01J 49/04 20060101
H01J049/04; C07K 14/195 20060101 C07K014/195 |
Claims
1. A method for expression of engineered constructs containing
sequences coding for functional genes of interest in E. coli
strains comprising: fusing genes of interest to an inducible,
active promoter to optimize transcription of the open reading
frames, and an introduced consensus Shine-Dalgarno sequence
allowing for optimal ribosome binding and translation of the open
reading frames, and codons optimized for expression in E. coil.
2. The method of claim 1 wherein the step of fusing includes
cloning tceA and tceB downstream of the arabinose-inducible
promoter in plasmid pBAD18.
3. A method for expression of engineered constructs containing
sequences coding for Reductive Dehalogenase enzymes (RDase) in E.
coli strains comprising: fusing genes of interest to an inducible,
active promoter to optimize transcription of the open reading
frames, and an introduced consensus Shine-Dalgarno sequence
allowing for optimal ribosome binding and translation of the open
reading frames, and codons optimized for expression in E. coil.
4. The method of claim 3 wherein the step of fusing includes
cloning tceA and tceB downstream of the arabinose-inducible
promoter in plasmid pBAD18.
5. A method for expression of engineered constructs containing
sequences coding for functional genes of interest in E. coli
strains comprising: fusing the genes of interest (i.e., tceA and
tceB) to an inducible, active promoter to optimize transcription of
the open reading frames, and an introduced consensus Shine-Dalgarno
sequence allowing for optimal ribosome binding and translation of
the open reading frames, and codons optimized for expression in E.
coil.
6. The method of claim 5 wherein the step of fusing includes
cloning tceA and tceB downstream of the arabinose-inducible
promoter in plasmid pBAD18.
7. An engineered construct containing sequences coding for
functional genes of interest in E. coli strains comprising: genes
of interest (i.e., tceA and tceB) fused to an inducible, active
promoter to optimize transcription of the open reading frames, and
an introduced consensus Shine-Dalgarno sequence allowing for
optimal ribosome binding and translation of the open reading
frames, and codons optimized for expression in E. coil.
8. The construct of claim 7 wherein tceA and tceB are cloned
downstream of the arabinose-inducible promoter in plasmid
pBAD18.
9. A method of overexpressing proteins originating in
Dehalococcoides or other difficult to grow anaerobic
microorganisms, with the method comprising: separating products of
gene expression, where the gene expression products include
proteins encoded on a pBAD18+gene of interest construct; and using
the separated proteins for de novo sequencing of peptides by
employing proteomic mass spectrometry.
10. The method of claim 9 wherein separating products of gene
expression comprises using SDS gel.
11. The method of claim 10 wherein the SDS gel comprises a 5-20%
SDS-polyacrylamide gel.
12. A mass spectrometric method for tracking the expression of
genes of Dehalococcoides or other difficult to grow anaerobic
microorganisms, comprising the steps of: acquiring an environmental
sample; extracting proteins and peptides contained therein;
introducing proteins and peptides originating from said sample into
a mass spectrometer; and analyzing the resultant mass spectra by
utilizing reference spectra from overexpressed proteins and
peptides from Dehalococcoides.
13. The method of claim 12 wherein the method further comprises
using labeled proteins and peptides for target quantification.
14. The method of claim 12 wherein the method further comprises
using isotope labeled peptides for protein and peptide
quantification.
15. A mass spectrometric method for tracking the expression of
genes of Dehalococcoides, comprising the steps of: acquiring an
environmental sample and extracting proteins and peptides contained
therein; introducing proteins and peptides originating from said
sample into a mass spectrometer; and monitoring ions of digestion
products by utilizing their characteristic mass-to-charge ratios
(m/z).
16. The method of claim 15 wherein the digestion products comprise
peptides.
17. A method of utilizing the metabolic capability of a living
organism, with the method comprising: (a) performing codon
optimization of the gene sequence to be expressed for the specific
expressing host; (b) using a transforming vector; (c) adding a
strong promoter and ribosomal binding sequences for increased
expression of the protein of interest; and (d) detecting the over
expressed protein using mass spectrometric methods.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Related technology is disclosed in pending PCT patent
application publication WO2011/011683, published on Jan. 27, 2011,
and entitled "MICROBIAL CULTURES AND METHODS FOR ANAEROBIC
BIOREMEDIATION," to co-inventors of the present application, of
which the entire content is incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to methods and systems for
tracking bioremediation processes. More particularly, the invention
relates to methods for the convenient expression of genes of
Dehalococcoides and other relevant microorganisms for remediation
and bioenergy and the mass spectrometric detection of gene
expression products for tracking the progress of bioremediation and
bioenergy related efforts during cleanup of
chloroethene-contaminanted environments.
[0003] The present invention shows an example of how this invention
is applied to detect and track a reductive dehalogenase enzyme in
Dehalococcoides i.e, TceA, however this over expression and
detection system can be applied to track many other enzymes of
interest.
BACKGROUND
[0004] Microorganisms of the family Dehalococcoides are known to
perform the beneficial biotransformation of toxic chlorinated
ethenes to non-toxic ethene at hazardous waste sites. At the
community level, Dehalococcoides performance can be improved by the
presence of homoacetogens, where the presence of methanogens
diverts electrons and slows down dechlorination. Quantification of
Dehalococcoides sp. using isolated DNA and quantitative PCR [1-2]
has become one of multiple lines of evidence for remediation
decision making; however, the information obtained based on these
DNA-based assays is limited. The current assays target 3 identified
RDases tceA [3], bvcA [4], and vcrA [5]. Two key limitations are:
1. The primers can be too specific and therefore may not fully
capture homolog proteins, which serve the same function but are
slightly different at the gene level, and 2. Quantifying
Dehalococcoides genes provides no information on microbial
interactions, and how these interactions affect dechlorination
rates.
[0005] The present invention overcomes these two limitations. The
disclosed methods and systems have the potential to enhance or even
replace existing procedures and materials for the determination of
bioremediation progress. The technology disclosed herein overcomes
existing limitations in the availability and production of proteins
for use as bioremediation agents and as diagnostic materials. The
instant over-expression method will allow development of more
specific and sensitive protein detection methods which are helpful
to detect Dehalococcoides enzymes but could also be applied to many
other systems.
BRIEF SUMMARY OF THE DISCLOSURE
[0006] This summary is provided to introduce, in a simplified form,
a selection of concepts that are further described below in the
Detailed Description. This summary is not intended to identify key
features of the claimed subject matter, nor is it intended to be
used as an aid in determining the scope of the claimed subject
matter.
[0007] A method of over-expressing proteins originating in
Dehalococcoides where gene expression products are separated (e.g.,
on an SDS gel), and where the gene expression products include
proteins encoded by a pBAD18 +a gene of interest, such as, for
example, a tceAB construct. The protein gels are refrigerated at
4.degree. C. and later used for de novo sequencing of peptides
using proteomic mass spectrometry.
[0008] In one aspect, the gel comprises a 5-20% SDS-polyacrylamide
gel.
[0009] In another aspect a mass spectrometric method for tracking
the expression of genes of Dehalococcoides includes the steps of
acquiring an environmental sample, extracting proteins and peptides
contained therein, introducing proteins and peptides originating
from said sample into a mass spectrometer, and analyzing the
resultant mass spectra by utilizing reference spectra from
overexpressed proteins and peptides from Dehalococcoides.
[0010] In another aspect the method is modified by the use of
labeled proteins and peptides for target quantification.
[0011] In another aspect the method is modified by using isotope
labeled peptides for (absolute) quantitation of target proteins and
peptides.
[0012] In another aspect, a mass spectrometric method for tracking
the expression of genes of Dehalococcoides includes acquiring an
environmental sample, extracting proteins and peptides contained
therein, introducing proteins and peptides originating from said
sample into a mass spectrometer, and monitoring ions featuring a
mass-to-charge ratio (m/z) specific to peptides resulting from
protein digestion with a suitable enzyme (e.g., trypsin) of
expression products of the target genes of interest (e.g., tceA
[3], bvcA [4], and vcrA).
[0013] In another aspect a method for expression of engineered
constructs containing sequences coding for functional genes of
interest in E. coli strains includes fusing tceA and tceB to an
inducible, active promoter to optimize transcription of the open
reading frames, and an introduced consensus Shine-Dalgarno sequence
allowing for optimal ribosome binding and translation of the open
reading frames, and codons optimized for expression in E. coil.
[0014] In another aspect, a method of utilizing the metabolic
capability of a living organism, with the method includes (a)
performing codon optimization of the gene sequence to be expressed
for the specific expressing host; (b) using a transforming vector;
(c) adding a strong promoter and ribosomal binding sequences for
increased expression of the protein of interest; and (d) detecting
the over expressed protein using mass spectrometric methods.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] While the novel features of the invention are set forth with
particularity in the appended claims, the invention, both as to
organization and content, will be better understood and
appreciated, along with other objects and features thereof, from
the following detailed description taken in conjunction with the
drawings, in which:
[0016] FIG. 1 schematically illustrates a prior art plasmid
construct map of the gene TceAB (50997-3) cloned in pBAD18 by EcoRI
and KpnI.
[0017] FIG. 2 schematically illustrates a pBAD18 cloning map.
[0018] FIG. 3 and FIG. 3A graphically illustrate the mass
spectrometric analysis of an over-expressed gene product, i.e., an
RDase (specifically TceA here), using Matrix Assisted Laser
Desorption Ionization/Tandem Time-Of-Flight mass spectrometry
(MALDI/TOF/TOF). The spectrum reveals specific peptides suitable
for mass spectrometric tracking by their characteristic
mass-to-charge ratios. To obtain the spectrum, the gene expression
product was digested with trypsin and analyzed by MALDI-TOF/TOF.
The resultant spectrum was analyzed with standard proteomics
software to reveal the identity and structure of peptides
corresponding to the mass-to-charge ratios observed.
[0019] In the drawings, identical reference numbers identify
similar elements or components. The sizes and relative positions of
elements in the drawings are not necessarily drawn to scale. For
example, the shapes of various elements and angles are not drawn to
scale, and some of these elements are arbitrarily enlarged and
positioned to improve drawing legibility. Further, the particular
shapes of the elements as drawn, are not intended to convey any
information regarding the actual shape of the particular elements,
and have been solely selected for ease of recognition in the
drawings.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0020] The following disclosure describes tracking bioremediation
processes. Several features of methods and systems in accordance
with example embodiments are set forth and described in the FIG.s.
It will be appreciated that methods and systems in accordance with
other example embodiments can include additional procedures or
features different than those shown in the FIG.s. Example
embodiments are described herein with respect to analysis of
environmental conditions. However, it will be understood that these
examples are for the purpose of illustrating the principles, and
that the invention is not so limited. Additionally, methods and
systems in accordance with several example embodiments may not
include all of the features shown in the FIG.s.
[0021] Unless the context requires otherwise, throughout the
specification and claims which follow, the word "comprise" and
variations thereof, such as, "comprises" and "comprising" are to be
construed in an open, inclusive sense that is as "including, but
not limited to."
[0022] Reference throughout this specification to "one example" or
"an example embodiment," "one embodiment," "an embodiment" or
combinations and/or variations of these terms means that a
particular feature, structure or characteristic described in
connection with the embodiment is included in at least one
embodiment of the present disclosure. Thus, the appearances of the
phrases "in one embodiment" or "in an embodiment" in various places
throughout this specification are not necessarily all referring to
the same embodiment. Furthermore, the particular features,
structures, or characteristics may be combined in any suitable
manner in one or more embodiments.
DEFINITIONS
[0023] Generally, as used herein, the following terms have the
following meanings when used within the context of contaminant
sample collection in aquatic or saturated sedimentary
environments:
[0024] A "sample" as used herein refers to material, such as
environmental material obtained from a remediation site that is
suspected of containing, or known to contain, an analytes, as for
example, proteins and peptides.
[0025] "EcoRI" is a well known endonuclease enzyme isolated from
strains of E. coli, and is part of the restriction modification
system used in cloning.
[0026] "KpnI" is a well known restriction enzyme.
[0027] The term "TOP10 strain" as used herein refers to E. coli
competent TOP10 strains as supplied for example, by Invitrogen
Corporation, now part of Life Technologies of California, US.
[0028] The term "TCE" as used herein refers to trichloroethene.
[0029] tceA is the gene that encodes for the enzyme TceA. TceA
performs the dechlorination (conversion to less chlorinated
compounds) of TCE.
[0030] RDase is a/any reductive dehalogenase, these are enzymes
that convert chlorinated compounds to less chlorinated
products.
EXAMPLE EMBODIMENTS
[0031] Referring now to FIG. 2, a pBAD18 cloning map is
schematically illustrated for designing, cloning and expression of
engineered constructs containing sequences coding for functional
genes of interest (in this case tceA and tceB) in E. coli strains.
A gene construct 10 contains optimal expression signals for a
functional tceA tceB cassette. The construct 10 comprises the
products of fusion of the above genes to an inducible, highly
active promoter to optimize transcription of the open reading
frames, and an introduced consensus Shine-Dalgarno sequence
allowing for optimal ribosome binding and translation of the open
reading frames, and codons optimized for expression in E. coil.
These features greatly enhance the ability to obtain enhanced
expression of the enzyme of interest, in this case the TceA and
TceB enzymes.
[0032] In one example, the 1792-bp section containing the two genes
of interest tceA and tceB was cloned downstream of the
arabinose-inducible promoter in plasmid pBAD18. The pBAD18 cloning
map includes an optimal Shine Dalgarno sequence followed by genes
tceA and tceB and a transcriptional terminator (15 in FIG. 2). The
codon optimization for TceAB over expression in E. coli is listed
as SEQ. NO. 1 in the appended sequence listing. The remaining DNA
sequences in the expression vector comprise the E. coli DNA for the
pBAD18 cloning vector sequence as detailed below and in the
appended sequence listing:
[0033] E. coli DNA for pBAD18 Cloning Vector (SEQ. NO. 2): [0034]
GenBank: X81838.1 [0035] LOCUS X81 838 461 3 by DNA circular SYN 25
Oct. 1995 [0036] DEFINITION E. coli DNA for pBAD18 cloning vector.
[0037] ACCESSION X81838 [0038] VERSION X81838.1 GI:551423 [0039]
KEYWORDS arac gene; bla gene; expression vector. [0040] SOURCE
synthetic construct [0041] ORGANISM synthetic construct [0042]
other sequences; artificial sequences. [0043] REFERENCE 1 (bases 1
to 4613) [0044] AUTHORS Guzman, L. M., Belin, D., Carson, M. J. and
Beckwith, J. [0045] TITLE Tight regulation, modulation, and
high-level expression by Vectors containing the arabinose PBAD
promoter [0046] JOURNAL J. Bacteriol. 177 (14), 4121-4130 (1995)
[0047] PUBMED 7608087 [0048] REFERENCE 2 (bases 1 to 4613) [0049]
AUTHORS Guzman, L. M. [0050] TITLE Direct Submission [0051] JOURNAL
Submitted (22-SEP-1994) L. M. Guzman, Harvard Medical School, Dept
of M Ave., Boston Mass. 02115, USA [0052] COMMENT Related
sequences: J01641 and M10196 (bases 1-1300); M77749 (bases
1357-2296; 4537-4613); SYNPBR322 (bases 2297-2780); Gene 27, 183,
1984 (bases 2780-3238).
[0053] Referring now to FIG. 3 and FIG. 3A, a confirmatory analysis
of successful TceA expression is shown here using Matrix Assisted
Laser Desorption Ionization/Tandem Time-Of-Flight (MALDI/TOF/TOF)
mass spectrometry. Gene expression products were separated on an
SDS gel. In one example, expression of the protein encoded on the
BAD18+tceAB construct was verified using a 12% SDS-polyacrylamide
gel. The protein gel was refrigerated at 4.degree. C. and later
used for de novo sequencing of peptides using proteomic mass
spectrometry. The corresponding band was extracted and analyzed by
peptide mass fingerprinting and peptide mass sequencing using
MALDI-TOF/TOF. This represents the first successful over-expression
of this TCE reductive dehalogenase (TCE-RDase). The disclosed
process demonstrates an ability to generate large quantities of key
enzymes of the slow-growing Dehalococcoides, a feat previously
unattained when using current state-of-the art methods.
[0054] The above over-expressed proteins were used to develop a
sensitive proteomic detection approach. Proteomic assays will make
use of isotope-labeled peptides to be custom-synthesized based on
the peptide mass fingerprints and ionization properties of digested
target RDases. These novel proteomic assays will employ the isotope
dilution technique for absolute quantitation of proteins in
environmental samples (AQUA). Using the capsid protein of the
Norovirus ("cruise ship virus") as a target, we have demonstrated
that the use of electrospray and MALDI tandem mass spectrometry
allows for the detection of attomole levels of target protein (1
atto mole=1.times.10.sup.-18 moles) [6].
[0055] The invention has been described herein in considerable
detail in order to comply with the Patent Statutes and to provide
those skilled in the art with the information needed to apply the
novel principles of the present invention, and to construct and use
such exemplary and specialized components as are required. However,
it is to be understood that the invention may be carried out by
different equipment, and devices, and that various modifications,
both as to the equipment details and operating procedures, may be
accomplished without departing from the true spirit and scope of
the present invention.
REFERENCES
[0056] The teachings of the following publications are incorporated
herein in their entirety by this reference. [0057] [1] Werner, J.
J., A. C. Ptak, B. G. Rahm, S. Zhang, and R. E. Richardson,
Absolute quantification of Dehalococcoides proteins: enzyme
bioindicators of chlorinated ethene dehalorespiration.
Environmental Microbiology, 2009. 11(10): p. 2687-2697. [0058] [2]
Ritalahti, K. M., B. K. Amos, Y. Sung, Q. Z. Wu, S. S. Koenigsberg
and F. E. Loffler (2006). "Quantitative PCR targeting 16S rRNA and
reductive dehalogenase genes simultaneously monitors multiple
Dehalococcoides strains." Applied and Environmental Microbiology
72(4): 2765-2774.
[0059] [3] Magnuson, J. K., M. F. Romine, D. R. Burris, and M. T.
Kingsley. 2000. Trichloroethene reductive dehalogenase from
Dehalococcoides ethenogenes: sequence of tceA and substrate range
characterization. Appl. Environ. Microbiol. 66:5141-5147.
[0060] [4] Krajmalnik-Brown R., T. Holscher, I. N. Thomson, F. M.
Saunders, K. M. Ritalahti, and F. E. Loffler. 2004 "Genetic
Identification of a Putative Vinyl Chloride Reductase in
Dehalococcoides sp. Strain BAV1". Applied and Environmental
Microbiology. 70(10): 6347-6351.
[0061] [5] Muller, J. A., B. M. Rosner, G. von Abendroth, G.
Meshulam-Simon, P. L. McCarty, and A. M. Spormann. 2004. Molecular
identification of the catabolic vinyl chloride reductase from
Dehalococcoides sp. strain VS and its environmental distribution.
Appl. Environ. Microbiol. 70:4880-4888.
[0062] [6] Colquhoun, D. R., Schwab, K. J., Cole, R. N., and R. U.
Halden. 2006. Detection of Norovirus Capsid Protein in Authentic
Standards and in Stool Extract by Matrix-Assisted Laser Desorption
Ionization and Nanospray Mass Spectrometry. Appl. Environ.
Microbiol. 72(4):2749-2755.
Sequence CWU 1
1
212018DNAArtificial SequenceRelated sequences J01641 and M10196
(bases 1-1300); M77749 (bases 1357-2296; 4537-4613); SYNPBR322
(bases 2297-2780); Gene 27, 183, 1984 (bases 2780-3238).
1tgggctagcg aattcaggag gacagctatg agtgaaaaat atcattcaac tgttacccgg
60cgtgatttta tgaaacgttt agggctggcc ggggccggcg ctggcgcact gggcgcggca
120gtgcttgcgg aaaacaactt accgcatgaa tttaaggatg tcgatgatct
gctgtcagcc 180ggtaaagcct tggagggtga tcacgctaat aaagtgaaca
accatccgtg gtgggtgacc 240acccgtgatc atgaagatcc gacgtgcaac
atcgattggt cgctgattaa acgttactcg 300ggttggaaca accaaggtgc
ctatttcctg ccggaagatt acttgagtcc aacttatacc 360ggcagacgtc
atacgattgt ggatagcaaa ttagaaattg aattacaggg taagaagtac
420cgtgattccg cgtttattaa aagtggtatc gactggatga aagagaacat
tgacccggat 480tatgatccag gggaactggg ctacggcgat cgccgtgaag
atgctcttat ctacgccgct 540actaacggtt cccacaattg ttgggaaaac
cctctttatg gccgctatga aggtagccgt 600ccgtacctga gtatgcgtac
catgaacggc attaatggcc tccatgaatt tggtcatgca 660gacatcaaga
cgactaacta cccgaaatgg gagggtacgc cggaagaaaa ccttctgata
720atgcggaccg cggcccgcta ttttggcgcc tcatccgtcg gtgctatcaa
gataaccgat 780aacgtgaaaa agattttcta tgccaaagtg caaccgtttt
gtctgggccc gtggtacacc 840atcaccaaca tggccgaata tattgaatat
ccggtgccgg ttgacaatta cgccattccg 900attgtgtttg aagacattcc
ggccgatcaa ggtcattatt cgtataagcg cttcggtggt 960gatgataaaa
ttgctgtgcc gaacgccctt gataacatat tcacgtacac gatcatgctg
1020ccagaaaaac gttttaaata cgcgcattcg atcccgatgg atccgtgttc
ttgcattgct 1080tacccgctgt tcacagaagt tgaagctagg attcagcaat
ttattgcggg cctgggttat 1140aattccatgg gtggcggggt tgaagcgtgg
gggccgggca gcgcttttgg aaacctatcg 1200gggctggggg aacagtcgcg
tgtcagcagc atcatcgagc cgcgttatgg atcaaatacc 1260aaaggttcac
tgcggatgct gacagacctg ccgctcgcac cgactaagcc aatcgacgcg
1320ggcatccgcg agttctgtaa aacttgcggc atctgcgcgg aacactgccc
gactcaagca 1380atctctcacg aaggcccgcg gtatgactct ccccattggg
attgtgttag cggttacgaa 1440ggttggcact tagattatca taaatgtatt
aactgcacca tctgtgaagc tgtttgccca 1500ttctttacca tgagtaataa
tagctgggtt cataatctgg tgaaaagcac cgtcgccact 1560acgccggttt
ttaatggctt tttcaaaaat atggaaggcg cgtttggcta tggtccccga
1620tatagtccga gccgtgatga gtggtgggct agtgaaaatc caattcgggg
cgcgagcgtt 1680gatatctttt aagagaaagg atggaataga ttattatggg
cggggctctg tattatttcc 1740tggttggaat gctgattggt ggtgccgcca
tttggtttat tacctatacc cagtttaaaa 1800atattagctt taagtggtgg
gaatggtctc tgatggcact gtcactgctg ctggtcagta 1860gcatctttca
acacatgtat tcttcaatga gcgtggaaat ggaatatcag agcgcattta
1920tgtatctggg cgtgtttggt actctggccg tgatcctaaa cctgatcgtg
tggcgtacgt 1980atagcggcag aaaagaataa aatggtaccg catgcaac
201824613DNAArtificial sequenceRelated sequences J01641 and M10196
(bases 1-1300); M77749 (bases 1357-2296; 4537-4613); SYNPBR322
(bases 2297-2780); Gene 27, 183, 1984 (bases 2780-3238).
2atcgatgcat aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac
60tccgtcaagc cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca
120ttcacttttt cttcacaacc ggcacggaac tcgctcgggc tggccccggt
gcatttttta 180aatacccgcg agaaatagag ttgatcgtca aaaccaacat
tgcgaccgac ggtggcgata 240ggcatccggg tggtgctcaa aagcagcttc
gcctggctga tacgttggtc ctcgcgccag 300cttaagacgc taatccctaa
ctgctggcgg aaaagatgtg acagacgcga cggcgacaag 360caaacatgct
gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg
420tactgacaag cctcgcgtac ccgattatcc atcggtggat ggagcgactc
gttaatcgct 480tccatgcgcc gcagtaacaa ttgctcaagc agatttatcg
ccagcagctc cgaatagcgc 540ccttcccctt gcccggcgtt aatgatttgc
ccaaacaggt cgctgaaatg cggctggtgc 600gcttcatccg ggcgaaagaa
ccccgtattg gcaaatattg acggccagtt aagccattca 660tgccagtagg
cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga
720tgacgaccgt agtgatgaat ctctcctggc gggaacagca aaatatcacc
cggtcggcaa 780acaaattctc gtccctgatt tttcaccacc ccctgaccgc
gaatggtgag attgagaata 840taacctttca ttcccagcgg tcggtcgata
aaaaaatcga gataaccgtt ggcctcaatc 900ggcgttaaac ccgccaccag
atgggcatta aacgagtatc ccggcagcag gggatcattt 960tgcgcttcag
ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat
1020tgcatcagac attgccgtca ctgcgtcttt tactggctct tctcgctaac
caaaccggta 1080accccgctta ttaaaagcat tctgtaacaa agcgggacca
aagccatgac aaaaacgcgt 1140aacaaaagtg tctataatca cggcagaaaa
gtccacattg attatttgca cggcgtcaca 1200ctttgctatg ccatagcatt
tttatccata agattagcgg atcctacctg acgcttttta 1260tcgcaactct
ctactgtttc tccatacccg tttttttggg ctagcgaatt cgagctcggt
1320acccggggat cctctagagt cgacctgcag gcatgcaagc ttggctgttt
tggcggatga 1380gagaagattt tcagcctgat acagattaaa tcagaacgca
gaagcggtct gataaaacag 1440aatttgcctg gcggcagtag cgcggtggtc
ccacctgacc ccatgccgaa ctcagaagtg 1500aaacgccgta gcgccgatgg
tagtgtgggg tctccccatg cgagagtagg gaactgccag 1560gcatcaaata
aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt
1620gtcggtgaac gctctcctga gtaggacaaa tccgccggga gcggatttga
acgttgcgaa 1680gcaacggccc ggagggtggc gggcaggacg cccgccataa
actgccaggc atcaaattaa 1740gcagaaggcc atcctgacgg atggcctttt
tgcgtttcta caaactcttt tgtttatttt 1800tctaaataca ttcaaatatg
tatccgctca tgagacaata accctgataa atgcttcaat 1860aatattgaaa
aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
1920ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
gtaaaagatg 1980ctgaagatca gttgggtgca cgagtgggtt acatcgaact
ggatctcaac agcggtaaga 2040tccttgagag ttttcgcccc gaagaacgtt
ttccaatgat gagcactttt aaagttctgc 2100tatgtggcgc ggtattatcc
cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac 2160actattctca
gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
2220gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
actgcggcca 2280acttacttct gacaacgatc ggaggaccga aggagctaac
cgcttttttg cacaacatgg 2340gggatcatgt aactcgcctt gatcgttggg
aaccggagct gaatgaagcc ataccaaacg 2400acgagcgtga caccacgatg
cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg 2460gcgaactact
tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
2520ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
gataaatctg 2580gagccggtga gcgtgggtct cgcggtatca ttgcagcact
ggggccagat ggtaagccct 2640cccgtatcgt agttatctac acgacgggga
gtcaggcaac tatggatgaa cgaaatagac 2700agatcgctga gataggtgcc
tcactgatta agcattggta actgtcagac caagtttact 2760catatatact
ttagattgat ttacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
2820gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
tcctttcgct 2880ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
gtcaagctct aaatcggggg 2940ctccctttag ggttccgatt tagtgcttta
cggcacctcg accccaaaaa acttgatttg 3000ggtgatggtt cacgtagtgg
gccatcgccc tgatagacgg tttttcgccc tttgacgttg 3060gagtccacgt
tctttaatag tggactcttg ttccaaactt gaacaacact caaccctatc
3120tcgggctatt cttttgattt ataagggatt ttgccgattt cggcctattg
gttaaaaaat 3180gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
tattaacgtt tacaatttaa 3240aaggatctag gtgaagatcc tttttgataa
tctcatgacc aaaatccctt aacgtgagtt 3300ttcgttccac tgagcgtcag
accccgtaga aaagatcaaa ggatcttctt gagatccttt 3360ttttctgcgc
gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
3420tttgccggat caagagctac caactctttt tccgaaggta actggcttca
gcagagcgca 3480gataccaaat actgtccttc tagtgtagcc gtagttaggc
caccacttca agaactctgt 3540agcaccgcct acatacctcg ctctgctaat
cctgttacca gtggctgctg ccagtggcga 3600taagtcgtgt cttaccgggt
tggactcaag acgatagtta ccggataagg cgcagcggtc 3660gggctgaacg
gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
3720gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
gaaaggcgga 3780caggtatccg gtaagcggca gggtcggaac aggagagcgc
acgagggagc ttccaggggg 3840aaacgcctgg tatctttata gtcctgtcgg
gtttcgccac ctctgacttg agcgtcgatt 3900tttgtgatgc tcgtcagggg
ggcggagcct atggaaaaac gccagcaacg cggccttttt 3960acggttcctg
gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
4020ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
gcagccgaac 4080gaccgagcgc agcgagtcag tgagcgagga agcggaagag
cgcctgatgc ggtattttct 4140ccttacgcat ctgtgcggta tttcacaccg
catatggtgc actctcagta caatctgctc 4200tgatgccgca tagttaagcc
agtatacact ccgctatcgc tacgtgactg ggtcatggct 4260gcgccccgac
acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
4320tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
gttttcaccg 4380tcatcaccga aacgcgcgag gcagcaagga gatggcgccc
aacagtcccc cggccacggg 4440gcctgccacc atacccacgc cgaaacaagc
gctcatgagc ccgaagtggc gagcccgatc 4500ttccccatcg gtgatgtcgg
cgatataggc gccagcaacc gcacctgtgg cgccggtgat 4560gccggccacg
atgcgtccgg cgtagaggat ctgctcatgt ttgacagctt atc 4613
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